Water-borne pathogens pose a serious threat to human health world-wide rendering water quality a fundamental determinant of the incidence and severity of disease. A critical factor in bacteria survival and proliferation in marine environments is the presence of grazing amoebae. These organisms feed on bacteria, playing a central role in restricting bacterial populations. However, many bacterial species have developed strategies to survive and replicate within amoebae. This has contributed to the emergence of human pathogens as these strategies can similarly be used by the bacterium to cause disease in humans. Amoebae also play an important role in the epidemiology of bacterial pathogens as their interaction enhances the pathogen's ability to cause an infection. Despite the importance of these interactions, little is known about the bacterial requirements for growth in amoebae. This severely limits our understanding of how these pathogens persist in environmental and man-made water sources and the factors that contribute to the incidence and severity of disease. The bacterial pathogen Legionella is present in over two thirds of potable water distribution systems. Exposure to this pathogen occurs through the inhalation of contaminated water aerosols which can result in life-threatening pneumonia. Legionella survives in potable water due to its ability to replicate within amoebae, which protects the bacterium from killing by water disinfection procedures. Confounding this problem, Legionella is able to grow in multiple types of amoebae. This is a key determinant of its survival in water reservoirs where amoebal populations are highly diverse. The goal of this research is to define bacterial requirements for growth in amoebae and the mechanisms responsible for the transmission of Legionella from environmental reservoirs to humans. To do this, high throughput phenotypic screening assays and functional genomics will be used to: 1) Systematically identify Legionella genes essential for replication in diverse amoebal hosts to define core virulence strategies commonly used in all hosts and auxiliary strategies that allow Legionella to adapt to variation between hosts; 2) Determine the conservation and functional importance of virulence genes across multiple environmental and clinical isolates to assess the utility of their encoded proteins as targets against a broad spectrum of Legionellae. These studies will provide a foundation for developing more effecting strategies for eliminating bacteria from water resources to reduce the risk of infection and unique insight into Legionella pathogenesis that will be instrumental in developing novel strategies for treating disease.